ABSTRACT ? Project 2, Nussenzweig and Rice Memory B cells are mediators of immunological memory and vaccine responses. Memory B cells are a heterogeneous group of cells, that can be distinguished based on phenotypic markers, function, location, the antibody isotype expressed, and the degree of antibody gene somatic mutation. Our understanding of memory B cell biology is based primarily on sophisticated experiments using genetically modified mice and model antigens. In contrast, we know much less about the human memory B cells, in particular about those that develop in response to a specific pathogen and during natural infection. To date most studies have focused on sequencing the antibodies from the memory B cells, while many basic aspects of their biology could not be studied in part due to the difficulty at identifying pathogen-specific cells and their relative paucity. Advances in single-cell technologies are starting to make these investigations possible. In Project 2, the Rice and Nussenzweig laboratories propose to work together to investigate and compare the B cell memory that develops in response to important human pathogens: the dengue and Zika flaviviruses (DENV and ZIKV) and the hepatitis B virus (HBV). These viruses are responsible for considerable morbidity and mortality. More than 40% of the world population lives in areas at risk for infection by DENV and ZIKV flaviviruses and about 250 million people are living with HBV infection worldwide, despite the existence of an efficacious HBV vaccine. The immune memory to these pathogens is interesting because it can be either protective (e.g. HBV vaccination) or potentially harmful (e.g. antibody dependent enhancement of infection with DENV). We hypothesize that the memory B cells elicited by DENV, ZIKV and HBV in selected individuals are distinct and characterized by the expression of genes that are either general or pathogen-specific and that are linked to features of the antibodies that they express. To test this hypothesis, we will combine antigen-specific B cell purification by cell sorting with single cell transcriptomics analysis. Our approach will take advantage of large cohorts of human samples obtained through ongoing collaborations with investigators in DENV and ZIKV endemic areas of Brazil and Mexico, as well as of HBV samples from naturally infected or vaccinated individuals obtained at the Rockefeller University Hospital. Samples from individuals with high DENV and ZIKV neutralizing activity from Brazil and Mexico will be identified and single memory B cells specific for DENV will be subjected to single cell RNA-seq to characterize their transcriptome and to obtain paired antibody heavy and light chain sequences (Aim 1). Similarly, we will analyze single memory B cells elicited by HBV infection or vaccination (Aim 2), and the memory transcriptomes of DENV, ZIKV and HBV will be compared to each other and to those of nave B cells. In Aims 3 and 4 we will clone and characterize the antibodies from the same cells, and link this information to the transcriptome and to the antibodies' ability to either protect (HBV) or enhance infection (DENV).